Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA’s Bacteriological Analytical Manual (BAM)

Authors: PARK, SEONG - Mississippi State University; ZHANG, YAN - Mississippi State University

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/4/2024
Publication Date: 9/7/2024
Citation: Park, S.B., Zhang, Y. 2024. Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA’s Bacteriological Analytical Manual (BAM). Pathogens. 13(9). https://doi.org/10.3390/pathogens13090774.
DOI: https://doi.org/10.3390/pathogens13090774

Interpretive Summary: Vibrio parahaemolyticus is a harmful bacterium that can cause serious foodborne illness when people eat contaminated seafood. To detect this bacterium and determine whether it is dangerous, laboratories often use a method recommended by the U.S. FDA that identifies three specific genes. However, the standard test has limitations as two of the gene signals are difficult to tell apart due to similar sizes, and one often appears weak during testing. This study introduces an improved version of the test by replacing one of the gene primers with a new version, making it easier to clearly detect all three gene markers. The improved method successfully separates the gene signals on a gel and enhances the visibility of all bands, making results easier to interpret. It also demonstrated high sensitivity, detecting very small amounts of bacterial DNA, and did not react to other Vibrio species or common foodborne pathogens. This updated test could offer seafood producers and food safety laboratories a more accurate and dependable tool for identifying V. parahaemolyticus and assessing its potential to cause illness.

Technical Abstract: Vibrio parahaemolyticus is an important foodborne bacterium that causes severe gastroenteritisfollowing the consumption of contaminated seafood. To identify V. parahaemolyticus and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)’s Bacteriological Analytical Manual (BAM) recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin (tlh) gene and the pathogenic thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the trh (486 bp) and tlh (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the tdh gene amplicon (270 bp). The present study aimed to improve the BAM’s multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the tlh gene (369 bp) alongside the existing primers for the trh and tdh genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of V. parahaemolyticus.